PROJECT SUMMARY Apolipoprotein E (APOE) is the most significant risk gene for late-onset Alzheimer's disease (AD). APOE ?4/?4 homozygosity increases AD risk by >14-fold. Although an association between the APOE ?4 allele and increased AD risk is well-established, the mechanisms underlying this genetic risk remain elusive. In brain, microglia and astrocytes induce inflammation and degrade (engulf, digest, store or recycle) lipid-rich cellular debris that accumulates during aging and neurodegeneration. We hypothesize that APOE ?4/?4 genotype has cell autonomous effects on astrocytes and microglia which affects other cell types. Specifically, we hypothesize that APOE ?4/?4 astrocytes and microglia exhibit altered response to lipid-rich debris (myelin fragments), detectable as changes in global transcription and cellular function. To test this hypothesis we have generated a unique series of isogenic iPSCs differing solely in APOE isoform using a CRISPR/Cas9 genome-editing tool. We have established a platform to recapitulate cellular systems of human brain in culture by differentiation of iPSC-derived astrocytes and microglia that express APOE. We seek to bring together this established patient- derived isogenic human iPSC model, powerful systems biology, bioinformatic approaches and in vitro metabolic assays including neuroinflammation, phagocytosis and autophagy to understand the mechanism underlying APOE risk for AD. This proposed research project sets out to unravel the APOE isoform-dependent effects in glia and their responses to lipid challenge. In aim 1, we will generate homogenous populations of astrocytes and microglia and mixed cultures of cortical neurons/glia from isogenic APOE isoforms and APOE knockout derived from patient iPSCs. We will perform differential gene expression analysis to determine the downstream effects of APOE genotype in each cell type. In aim 2, we will study APOE isoform-dependent glial responses to challenge with lipid-rich particles. Following an unbiased transcriptomic approach, we will analyze which of the disease associated microglia signatures or AD-associated networks are APOE isoform-dependent upon challenge. In aim 3, we will investigate APOE isoform-dependent effects on the inflammatory response and phagocytic/autolysosomal clearance of lipid particles. The goal of this project is to identify the transcriptomic networks and cellular functions governed by APOE genotype in the presence or absence of a disease relevant environment (myelin debris) to pinpoint the earliest and potentially most treatable mechanisms involved in AD pathogenesis.